The only enzyme required by the bacteriophage φ29 to replicate its genome is its DNA polymerase, a 66 KDa monomeric protein capable of catalyzing both the initiation of the replication and the elongation of the synthesized strand. For the initiation, this polymerase is bound to a protein known as “terminal” (TP), recognizes the end of the φ29 DNA and catalyzes the formation of a TP-dAMP covalent complex. After the polymerization of 10 nucleotides, the DNA polymerase/TP heterodimer disassociates and the elongation of the strand coming from DNA is carried out.
Replicative DNA polymerases require the interaction with accessory proteins which stabilize the binding between the enzyme and the DNA (Kuriyan and O'Donnell. J Mol Biol. 1993; 234: 915-925). On the other hand, said DNA polymerases need to couple the polymerization upon the detachment of the DNA strand which is not being copied for which they require the functional association thereof to helicase type proteins. In this sense, the DNA polymerase of the bacteriophage φ29 has various intrinsic functional characteristics making it unique:                a) High processivity (defined as the number of nucleotides incorporated by binding event).        b) High strand detachment capacity which allows replicating the genome of said bacteriophage in the absence of helicase type accessory proteins. These two characteristics, processivity and strand detachment allow the φ29 DNA polymerase to be capable of synthesizing DNA strands of more than 70 kb in length (Blanco et al. J Biol Chem. 1989; 264: 8935-8940).        c) High accuracy in the insertion of nucleotides in the new strand (Esteban et al. J Biol Chem. 1993; 268: 2719-2726).        
All these characteristics have led to the development of a great variety of isothermal process (at constant temperature) protocols for amplifying double stranded DNA (dsDNA) based on the use of this polymerase. In a simple configuration, the capacity of the φ29 DNA polymerase to use circular single stranded DNA (ssDNA) allows amplifying DNA by the rolling circle method (or RCA—rolling-circle amplification), producing ssDNA molecules of great length and containing more than 10 copies of the circular template (Blanco et al. J Biol Chem. 1989; 264: 8935-8940; U.S. Pat. No. 5,001,050, U.S. Pat. No. 5,198,543 and U.S. Pat. No. 5,576,204). In the process for amplifying dsDNA developed by Amersham Biosciences/Molecular Staging (Dean et al. Genome Res. 2001; 11: 1095-1099; Dean et al. Proc Natl Acad Sci USA. 2002; 99: 5261-5266), the combination of the use of the φ29 DNA polymerase with the use of hexamers (hexa-nucleotides) random sequence primers allows obtaining amplification factors of 104-106 starting from picograms of circular plasmid DNA [Templiphi™ of GE Healthcare] or from 10 nanograms of Genomic DNA [Genomiphi™ of GE Healthcare and Repli-G® of Qiagen]. The products produced are of high quality and can be digested or sequenced directly without the need of prior purification, it has been demonstrated that the φ29 DNA polymerase is the most robust enzyme for this purpose. The common buffer for carrying out the amplification reactions with the φ29 DNA polymerase contains tris-HCl (pH 7.5) plus different concentrations (in the millimolar order) of NaCl or KCl and MgCl2 (US20030207267). However, in spite of the satisfactoriness of these protocols in very diverse situations, the development of other protocols which allow starting from lesser DNA amounts is a growing need.
The HhH (“helix-hairpin-helix”) motifs bind the DNA regardless of its sequence and are found in various DNA polymerases, ligases and glycosylases (Shao and Grishin. Nucleic Acids Res. 2000; 28: 2643-2650; Doherty et al. Nucleic Acids Res. 1996; 24:2488-2497). These motifs contain a pair of anti-parallel α-helixes connected by a “hairpin” type loop. The second α-helix does not protrude from the structure and therefore, unlike other DNA binding motifs, it cannot be intercalated in the major groove of the DNA. Crystallographic studies suggest that the protein-DNA interactions are established through the “loop” between the two α-helixes. This loop is involved in establishing nonspecific interactions with the DNA and normally contains the consensus sequence GhG, wherein h is a hydrophobic residue normally I, V, or L. The resolution of crystallographic structures suggests that the interactions are established between the nitrogen of the polypeptide strand and the oxygen of the phosphates of the DNA. Furthermore, polar amino acids which would establish additional interactions with the phosphate groups tend to exist in the positions 2 and 3 with respect to the second G. The last G of the consensus sequence forms the N-terminal part of the second α-helix and the hydrophobic residue h contributes to the interactions between the two α-helixes of the motif. The two α-helixes are packaged forming an angle of 25-50° between one another dictating the characteristic hydrophobic pattern in the sequences. The HhH motifs generally form part of major structures known as (HhH)2 made up by two HhH motifs bound by an α-helix, forming a mirror symmetry with respect to that of the DNA facilitating the stable binding thereof to the same (Shao and Grishin. Nucleic Acids Res. 2000; 28: 2643-2650; Doherty et al. Nucleic Acids Res. 1996; 24:2488-2497; Thayer et al. EMBO J. 1995; 14: 4108-4120).
The formation of chimeras between heat stable DNA polymerases such as Taq and Pfu and nonspecific DNA binding motifs has been previously used to increase the DNA binding capacity by said polymerases. (Pavlov et al. Proc Natl Acad Sci USA. 2002; 99: 13510-13515; WO2004013279; Wang et al. Nucleic Acids Res. 2004; 32: 1197-1207).
The crystallographic resolution of the structure of the φ29 DNA polymerase has provided the molecular bases responsible for the processive polymerization coupled to the strand detachment, a specific characteristic of this enzyme (Kamtekar et al. 2006; EMBO J 25: 1335-1343).
The comparative analysis with other DNA polymerases of the eukaryotic type (family B) shows a similar general folding: a C-terminal polymerization domain formed by the universal subdomains fingers, palm and thumb and forming a channel through which the DNA is bound; and a 3″-5″ N-terminal exonuclease domain responsible for removing the nucleotides mistakenly incorporated during the polymerization. The main structural difference between the DNA polymerases of known structure and that of φ29 is the presence, in the latter, of two additional subdomains in its polymerization domain, both corresponding to insertions of conserved sequence in the subgroup of the DNA polymerases using a protein called TPR1 and TPR2 as primer. The subdomain TPR1 is located close to palm and contacts with the DNA duplex. The subdomain TPR2, with a β-hairpin structure forms, close to the subdomains thumb, palm and fingers, an annular structure which would completely surround the newly synthesized DNA, binding the DNA polymerase to the DNA, required for replicating in a processive manner. Likewise, the subdomain TPR2, together with the subdomains fingers, palm and the exonuclease domain, participates in the formation of a narrow channel through which the template strand passes to access the active center during replication, forcing the separation of the double stranded DNA as the polymerase is displaced, acting in a manner similar to how a helicase would act and providing the polymerase with its capacity of coupling the polymerization to the strand detachment (Kamtekar et al. 2006; EMBO J 25: 1335-1343; Rodriguez et al. 2005; Proc Natl Acad Sci USA 102: 6407-6412). Such significant differences in the polymerization domain of the φ29 DNA polymerase with respect to the rest has the effect that the fusion of a peptide in the C-terminal end thereof would have unpredictable binding properties thereof in the polymerization and DNA.